Endiandric acid H and its derivatives, process for their preparation and use thereof

ABSTRACT

The invention relates to compounds of the formula (I)  
                 
 
     process for their preparation starting from the plant  Beilschmiedia fulva,  PLA 101 037, and the use thereof for producing a medicament, in particular for the treatment of allergic disorders, of asthmatic disorders, of inflammatory concomitant symptoms of asthma and/or of diseases which can be treated by inhibiting c-maf and NFAT.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/438,283 filed Jan. 6, 2003, and right of priorityfrom German Patent Application No. 10235624.6, filed Aug. 2, 2002.

[0002] Asthma is a disorder of the immune system which is manifested forexample as bronchial asthma in the form of episodes of acutely occurringshortness of breath with expiratory ventilation obstruction. The onlymedicaments available to date for treating asthma are those whichalleviate the symptoms, but none which intervene to inhibit themechanism responsible for the expression of mediators of inflammation.These mediators of inflammation, especially the cytokines interleukin-4(IL-4), interleukin-13 (IL-13) and interleukin-5 (IL-5) areoverexpressed in the asthmatic disorder and are responsible forinflammation, eosinophilia, mucus formation and bronchialhyperreactivity, which maintain the disease. The expression of thesecytokines is increased in the lungs of asthmatics (Ray & Cohn, J. Clin.Invest. 1999, 104(8), 985-993). Studies with transgenic animals showedthat a knockout of the IL-4 gene reduces the allergic inflammatoryreactions. Kim et al. (Immunity 1999, 10, 745) has shown that thetranscription factor of c-maf is responsible for the tissue-specificexpression of IL-4 in a subclass of T-helper cells and thus for allergicinflammatory reactions. c-maf belongs to the Maf family, a family ofleucine-containing zipper proteins, which is involved in the regulationof the expression of a whole series of genes. In TH2 cells, a subgroupof CD4+ helper cells, c-maf brings about, in synergy with NFAT (nuclearfactor of activated T cells), a transactivation of the IL-4 promoter,which in turn leads to an increase in the cytokine concentration.

[0003] An inhibitor of c-maf ought therefore to reduce the cytokineconcentration, resulting in a lower IL-4 level, which leads to a reducedIgE (immunoglobulin E) concentration because IL-4 is crucial for thestimulation of B cells to produce IgE. A reduction in IgE in turn wouldresult in a reduced mast cell degranulation and a reduced release ofhistamine, serotonin and other inflammatory factors. On the other hand,Ho et al. (J. Exp. Med., 1998,10, 1859-1866) describe regulation of theconcentration of the TH2 phenotype by IL-4 in an autocrine pathway. Areduction in the IL-4 level therefore results in a reduction in thisT-cell type, which in turn shifts the equilibrium between TH1 and TH2 inthe direction of TH1. This would further reduce the IL-4 concentrationand would have the additional advantage of reducing the IL-5 or IL-13production, which would result in a reduction in the eosinophilia, mucusformation and bronchial hyperreactivity.

[0004] Genes selectively expressed in TH2 cells are therefore preferredtargets for a therapeutic application for selectively influencing theinflammatory component in asthma and allergy.

[0005] In this connection, IL-4 and IL-5 antibodies are currently beingtested in clinical study (Foster et al., Trends Mol. Med., 2002, 8,162).It is expected that selective therapeutic agents will replace thewidespread therapy with glucocorticoids, which has to date representedthe only possibility for controlling the inflammatory reactions inasthma.

[0006] A tetracyclic compound of the formula

[0007] in which n is 0 is referred to as endiandric acid A, and in whichn is 1 as endiandric acid B. The compounds can be obtained from extractsof the Lauraceae family of plants, specifically of the genera Endiandraand Beilschmiedia (Bandaranayake et al., Aust. J. Chem., 1981,34,1655-67; Banfield et al., Aust. J. Chem., 1994, 47, 587-607).

[0008] Bandaranayake et al. describe the dihydro and tetrahydroderivatives of endiandric acid A, obtainable by partial or completereaction with hydrogen in the presence of Pd/C.

[0009] Banfield et al. describe endiandric acid A substituted on thephenyl radical by a methylenedioxy group.

[0010] Also known are derivatives of endiandric acid A and B in whichthe acid group is refunctionalized, for example as CH₂OSiPh₂t-Bu, CH₂Br,CH₂CN, esterified to CO₂CH₃, or reduced to the aldehyde CHO or to thealcohol CH₂OH (Nicolaou et al., J. Am. Chem. Soc., 1982,104, 5555-5557and 5560-5562).

[0011] Endiandric acid derivatives in which the double bond in the ringadjacent to the phenyl ring is in conjugation with the phenyl ring havenot previously been disclosed. Bandaranayake et al. describe the failureof corresponding isomerization attempts.

[0012] The biological effect of endiandric acid derivatives has notpreviously been investigated.

[0013] It is an object of the present invention to provide novelendiandric acid derivatives.

[0014] It has been found, surprisingly, that the African plantBeilschmiedia fulva is able to produce highly active novel compoundswhich are active as c-maf inhibitors.

[0015] The present invention therefore relates to a compound of theformula (I)

[0016] where

[0017] R₁ and R₂ are, independently of one another,

[0018] 0.0 H or

[0019] 1.0 a —O—C₁-C₆-alkyl, —O—C₂-C₆-alkenyl, —O—C₂-C₆-alkynyl or—O—C₆-C₁₀-aryl group, in which alkyl, alkenyl and alkynyl arestraight-chain or branched, and in which the alkyl, alkenyl and alkynylgroups are optionally mono- or disubstituted by:

[0020] 2.1 —OH

[0021] 2.2 ═O,

[0022] 2.3 —O—C₁-C₆-alkyl in which alkyl is straight-chain or branched,

[0023] 2.4 —O—C₂-C₆-alkenyl in which alkenyl is straight-chain orbranched,

[0024] 2.5 —C₆-C₁₀-aryl,

[0025] 2.6 —NH—C₁-C₆-alkyl in which alkyl is straight-chain or branched,

[0026] 2.7 —NH—C₂-C₆-alkenyl in which alkenyl is straight-chain orbranched,

[0027] 2.8 —NH₂ or

[0028] 2.9 halogen,

[0029] and in which the aryl groups are optionally mono- ordisubstituted by substituents 2.1 or 2.3 to 2.9,

[0030] in which the substituents 2.3, 2.4, 2.6 and 2.7 may be furthersubstituted by —CN, -amide or -oxime functions, and 2.5 may be furthersubstituted by —CN or amide functions or

[0031] R₁ and R₂ together form a ring, in which case R₁ and R₂ are agroup —O—[(C₁-C₆)-alkylene]—O—,

[0032] R₃ is

[0033] 1.0 H or

[0034] 2.0 a C₁-C₆-alkyl, C₂-C₆-alkenyl, C₂-C₆-alkynyl or C₆-C₁ ₀-arylgroup, in which alkyl, alkenyl and alkynyl are straight-chain orbranched, and in which the alkyl, alkenyl and alkynyl groups areoptionally mono- or disubstituted by:

[0035] 2.1 —OH,

[0036] 2.2 ═O,

[0037] 2.3 —O—C₁-C₆-alkyl in which alkyl is straight-chain or branched,

[0038] 2.4 —O—C₂-C₆-alkenyl in which alkenyl is straight-chain orbranched,

[0039] 2.5 —C₆-C₁₀-aryl,

[0040] 2.6 —NH—C₁-C₆-alkyl in which alkyl is straight-chain or branched,

[0041] 2.7 —NH—C₂-C₆-alkenyl in which alkenyl is straight-chain orbranched,

[0042] 2.8 —NH₂ or

[0043] 2.9 halogen,

[0044] and in which the aryl groups are optionally mono- ordisubstituted by substituents 2.1 or 2.3 to 2.9,

[0045] in which the substituents 2.3, 2.4, 2.6 and 2.7 may be furthersubstituted by —CN, -amide or -oxime functions, and 2.5 may be furthersubstituted by —CN or amide functions, and

[0046] R₄ is

[0047] CO₂R₃, CO₂NHR₃, CHO, CH₂OR₃, CH₂OSi(R₃)₃, CH₂Br, CH₂CN, where R₃is as defined above,

[0048] or a stereoisomeric form of the compound of the formula (I) or aphysiologically tolerated salt of the compound of the formula (I) or asalt of a stereoisomeric form of the compound of the formula (I).

[0049] means independently of one another a single bond or a doublebond.

[0050] The invention preferably relates to a compound of the formula(II)

[0051] where the radicals R₁ to R₄ are as defined above.

[0052] As used herein, “C₁-C₆-Alkyl” is a straight-chain or branchedalkyl having 1 to 6 carbon atoms, preferably having 1 to 4 carbon atoms,e.g. methyl, ethyl, i-propyl, tert-butyl and hexyl.

[0053] As used herein, “C₂-C₆-Alkenyl” is a straight-chain or branchedalkenyl having 2 to 6 carbon atoms, which is mono-, di- ortriunsaturated, e.g. allyl, crotyl, 1-propenyl, penta-1,3-dienyl andpentenyl.

[0054] As used herein, “C₂-C₆-Alkynyl” is a straight-chain or branchedalkynyl having 2 to 6 carbon atoms, which is mono- or diunsaturated,e.g. propynyl, butynyl and pentynyl.

[0055] As used herein, “C₆-C₁₀-Aryl” is an aryl group having 6 to 10carbon atoms, e.g. phenyl, benzyl or 1- or 2-naphthyl, which may also besubstituted, for example by halogen such as chlorine, bromine, fluorine,by alkyl having 1-4 carbon atoms, preferably methyl, by hydroxyl, byalkoxy having 1-4 carbon atoms, in particular methoxy, or bytrifluoromethyl.

[0056] As used herein, “C₁-C₆-Alkylene” means an alkylene group having 1to 6 carbon atoms, preferably having 1 to 4 carbon atoms, e.g.methylene, ethylene, i-propylene, tert-butylene and hexylene.

[0057] As used herein,“treat” or “treating” means any treatment,including but not limited to, alleviating symptoms, eliminating thecausation of the symptoms either on a temporary or permanent basis, orto preventing or slowing the appearance of symptoms and progression ofthe named disease, disorder or condition.

[0058] R₁ and R₂ are preferably H or a group—O—[(C₁-C₆)-alkyl]-O—,particularly preferably —O—CH₂—O—.

[0059] R₃ is preferably H.

[0060] R₄ is preferably COOR₃, where R₃ preferably H.

[0061] The invention particularly preferred relates to a compound of theformula (III)

[0062] The invention particularly most preferred relates to a compoundof the formula (IV), which is referred to hereinafter as endiandric acidH:

[0063] The compounds of the invention of the formula (I) contain atetracyclic ring system with a phenyl radical which, in contrast to thecompounds described previously, is in conjugation with one of the twodouble bonds in the tetracyclic ring system, and have an additionalhydroxyl group. Because of their different chemical structure, thecompounds of the formula (I) have novel physicochemical, biological andpharmacological properties.

[0064] The invention further relates to a compound obtainable from theplant Beilschmiedia fulva, PLA 101037, or cell cultures of the plantBeilschmiedia fulva, PLA 101037, characterized by the molecular formulaC₂₂H₂₁O₅, by the ¹H-NMR chemical shifts δ=1.733, 1.801, 1.209, 1.851,1.946, 2.339, 2.354, 2.400 2.440, 2.765, 3.351, 4.000, 5.207, 5.593,5.845, 6.00, 6.01, 6.873, 6.908, 6.995 and by the ¹³C-NMR chemicalshifts δ=33.62, 33.76, 34.82, 34.99, 40.32, 40.61, 40.69, 40.90, 44.02,71.96, 100.92, 106.66, 108.10, 119.86, 126.33, 127.59, 131.41, 133.76,140.15, 146.48, 147.51 173.47.

[0065] The invention further relates to a process for preparing thecompound of the formula (I), which comprises

[0066] 1. extracting the plant Beilschmiedia fulva, PLA 101037, or cellcultures of the plant Beilschmiedia fulva, PLA 101037, under suitableconditions,

[0067] 2. isolating the compound of the formula (IV), and

[0068] 3. where appropriate derivatizing to a compound of the formula(I) and/or reacting to give a physiologically tolerated salt of thecompound of the formula (I).

[0069] The invention further relates to a process for preparing acompound of the formula (IV), which comprises

[0070] 1. extracting the plant Beilschmiedia fulva, PLA 101037, or cellcultures of the plant Beilschmiedia fulva, PLA 101037, under suitableconditions,

[0071] 2. isolating the compound of the formula (IV), and

[0072] 3. where appropriate reacting to give a physiologically toleratedsalt of the compound of the formula (IV).

[0073] For the extraction of the compound of the formula (IV) from theplant Beilschmiedia fulva or one of its variants or mutants, the latteris initially cultivated under suitable conditions until endiandric acidH of the formula (IV) accumulates in the plant material. The plantBeilschmiedia fulva, its mutants and/or variants is preferablycultivated on suitable soils in a tropical or subtropical climate.Production of the plants is particularly preferably carried out undertropical conditions, for example at a temperature between 18 and 35° C.and at a humidity of greater than or equal to 70%, preferably of 70-90%.

[0074] To extract the compound of the formula (IV) from living cells ofthe plant Beilschmiedia fulva, the latter are initially transferred intoa suitable nutrient solution and cultivated until the compound of theinvention of the formula (IV) accumulates in the medium. The cellcultures are preferably set up as callus cultures. The nutrient mediaconsist, besides minerals and vitamins, also of at least one carbonsource, for example sucrose, and at least one nitrogen source, such as,for example, a nitrate or ammonium salt.

[0075]Beilschmiedia fulva is a tree from the Lauraceae family. TheLauraceae family includes many evergreen tropical spice plants anduseful plants. The geographical range of the Lauraceae covers the entiretropics: the sample of the plant Beilschmiedia fulva, PLA 101037, wascollected in Gabon, specifically in the area around la Makandé in theregion of the canopy of the rainforest. The sample was collected in thedirect vicinity of the Mankandé Research Field Station (coordinates 0°40′ 860″ S-11° 54′ 750″ E). Beilschmiedia fulva is a tree which mayreach a height of 30 m and thus forms part of the canopy region of therainforest. The leaves are bluish green, and the fruit is red. The trunkis cylindrical at the base and is sparsely covered with bark of brittleconsistency and has a brownish-reddish color. The bark layer is about4-5 mm thick and has a characteristic odor.

[0076] It is also possible to use other species from the genusBeilschmiedia, or else plants of the same species derived from adifferent site, to isolate endiandric acid H. The content of endiandricacid H may vary depending on the site conditions such as, for examplesoil characteristics, temperature, moisture, light incidence.

[0077] The process of the invention can be employed for extraction andisolation in a wide range of plant material to be extracted, for exampleon the laboratory scale (100 g to 1 kg of dried plant material) up tothe industrial scale (100 to >1000 kg).

[0078] The plant Beilschmiedia fulva can be cultivated outside or,preferably, in a greenhouse.

[0079] An alternative possibility is to employ cell cultures ofBeilschmiedia fulva plants to produce the compounds of the invention.This is normally done by cultivation in a plurality of stages, i.e.firstly one or more precultures are produced in a suitable liquid mediumand can then be used to inoculate the main culture. The startingmaterial is usually callus cultures. It is possible by choosing suitablebioreactors for growing the plant cell culture to achieve optimal mixingand aeration of the culture without the plant cells being exposed toexcessive shear forces, and thus optimal cell growth and metaboliteproduction. It is possible to employ for example airlift or bubblecolumn reactors, and paddle or propeller stirrers for mixing thecultures. The cells may grow as single cells or branched or unbranchedcell aggregates or chains. Metabolite production can be induced bystimulation with exogenous factors, e.g. heavy metal salts or plantelicitors.

[0080] Product formation in the plant cell culture can be monitored bymeans of the pH of the cultures and by chromatographic methods such as,for example, thin layer chromatography, HPLC or testing the biologicalactivity. Endiandric acid H of the formula (IV) may be present, besidesthe bark, also in other parts of the plant.

[0081] The endiandric acid H of the invention of the formula (IV) isisolated and purified from the plant or the culture medium by knownmethods which take account of the chemical, physical and biologicalproperties of the natural products. HPLC can be used to assay theconcentration of the compounds of the invention in the starting materialor in the individual stages of isolation, it being expedient to comparethe amount of substance formed with a calibration solution.

[0082] To isolate the endiandric acid H of the invention of the formula(IV), the Beilschmiedia fulva plant is harvested, initially collectingthe leaves, stems, wood, the bark or roots and separating according toparts of the plant, while still in the fresh state or dry, and thenextracting from the plant material with an organic solvent whichcontains water where appropriate. The compounds of the invention arepreferably isolated by extracting the bark.

[0083] The extracts are combined, diluted with water and extracted witha suitable, water-immiscible organic solvent, for example withn-butanol. The organic phase which has subsequently been separated offis concentrated where appropriate in vacuo. Fats can be removed from therequired product by diluting the concentrate with a nonpolar solvent inwhich the endiandric acid H derivative according to the invention isvery slightly soluble, such as, for example, with hexane, petroleumether, diethyl ether. This entails precipitation of the endiandric acidH, and the lipophilic impurities remain dissolved and are removed byconventional solid/liquid phase separations. The precipitate containingthe endiandric acid H is lyophilized. The lyophilizate is purifiedfurther.

[0084] Further purification of endiandric acid H takes place bychromatography on suitable materials, preferably, for example, onmolecular sieves, on normal phase supports such as, for example, silicagel, alumina, on ion exchangers or on adsorber resins, or on reversephases (reversed phase, RP). The endiandric acid H is removed by meansof this chromatography. The chromatography of the endiandric acid Htakes place with buffered aqueous solutions or mixtures of aqueous andorganic solutions.

[0085] Mixtures of aqueous and organic solutions mean all water-miscibleorganic solvents, preferably methanol, 2-propanol and acetonitrile, in aconcentration of from 10 to 90% of solvent, preferably 15 to 60% ofsolvent, or else all buffered aqueous solutions which are miscible withorganic solvents.

[0086] The endiandric acid H is removed with the aid of reversed phasechromatography, for example on MCI® (adsorber resin from Mitsubishi,Japan) or Amberlite XAD® (TOSOHAAS), on other hydrophobic materials suchas, for example, on RP-8 or RP-18 phases or on polyamides. Separation isadditionally possible with the aid of gel chromatography or normal phasechromatography, for example on silica gel or alumina.

[0087] The chromatography of endiandric acid H takes place with bufferedor acidified aqueous solutions or mixtures of aqueous solutions withalcohols or other water-miscible organic solvents. Propanol andacetonitrile is preferably used as organic solvent.

[0088] Buffered or acidified aqueous solutions mean, for example, water,phosphate buffer, citrate buffer, ammonium acetate in a concentration offrom 1 mM to 0.5 M, preferably 10 mM, and formic acid, acetic acid,trifluoroacetic acid or all commercially available acids known to theskilled worker, preferably in a concentration of from 0.01 to 3%.

[0089] Chromatography is carried out with a gradient which starts with100% aqueous buffer and finishes with 100% solvent, preferably running alinear gradient from 10 to 60% 2-propanol or acetonitrile.

[0090] The gel chromatography is carried out on polyacrylamide orcopolymer gels such as, for example, Biogel-P 2® (from Biorad),Fractogel TSK HW 40® (from Merck, Germany or Toso Haas, USA) or onSephadex® (Pharmacia, Uppsala, Sweden).

[0091] The sequence of the aforementioned chromatographies may bereversed.

[0092] A further purification step for the compounds of the invention iscrystallization. For example, endiandric acid H of the formula (IV)crystallizes from organic solvents and from mixtures of water withorganic solvents. The crystallization is carried out in a manner knownper se for example by concentrating or cooling saturated solutions.

[0093] Endiandric acid H of the formula (IV) is stable in the solidstate and in solutions in the pH range between 3 and 8, in particular 4and 6.

[0094] The compound of the formula (I) can be derivatized by methodsknown per se (J. March, Advanced Organic Synthesis, 4th Edition, JohnWiley & sons., 1992). For example, the carboxyl function can beesterified or reduced to the primary alcohol, or it can be convertedinto an amide. Carbonyl groups can be reduced with metal hydrides suchas aluminum hydrides or boron hydrides. Reduction to the saturatedcompounds can be achieved for example with hydrogen in the presence ofsuitable catalysts. The hydroxyl function can be esterified.

[0095] Compounds of the formula (I) can be converted into theirphysiologically tolerated salts by methods known to the skilled worker.

[0096] Physiologically tolerated salts of compounds of the formula (I)mean both their organic and inorganic salts as described in Remington'sPharmaceutical Sciences (17th edition, page 1418 (1985)). Because of thephysical and chemical stability and the solubility, sodium, potassium,calcium and ammonium salts are preferred, inter alia, for acidic groups.Preferred for basic groups are, inter alia, salts of hydrochloric acid,sulfuric acid, phosphoric acid or of carboxylic acids or sulfonic acids,such as, for example, acetic acid, citric acid, benzoic acid, maleicacid, fumaric acid, tartaric acid and p-toluenesulfonic acid.

[0097] The present invention includes all stereoisomeric forms of thecompounds of the formula (I). All possible enantiomers anddiastereomers, as well as mixtures of two or more stereoisomeric forms,for example mixtures of enantiomers and/or diastereomers, in all ratios,belong to the invention. The invention relates to enantiomers inenantiopure form, both as levorotatory and as dextrorotatory antipodes,R and S configurations, in the form of racemates and in the form ofmixtures of the two enantiomers in all ratios. If a cis/trans isomerismexists, the invention relates both to the cis form and to the trans formand to mixtures of these forms in all ratios.

[0098] Because of the pharmacological properties, the compounds of theinvention of the formula (I) are suitable for use as medicaments inhuman and/or veterinary medicine.

[0099] The invention therefore relates to a medicament having a contentof at least one compound of the formula (I) and of one or morephysiologically suitable excipients.

[0100] The medicaments of the invention are generally administeredorally, locally or parenterally, but rectal use is also possible inprinciple. Examples of suitable solid or liquid pharmaceutical forms aregranules, powders, tablets, coated tablets, (micro)-capsules,suppositories, syrups, emulsions, suspensions, aerosols, drops orinjectable solutions in ampul form, and products with protracted releaseof active ingredient, in the production of which normallyphysiologically suitable aids such as carriers, disintegrants, binders,coating agents, swelling agents, glidants or lubricants, flavorings,sweeteners or solubilizers are used. Carriers or excipients which arefrequently used and which may be mentioned are, for example, magnesiumcarbonate, titanium dioxide, lactose, mannitol and other sugars, talc,milk protein, gelatin, starch, vitamins, cellulose and its derivatives,animal or vegetable oils, polyethylene glycols and solvents such as, forexample, sterile water, alcohols, glycerol and polyhydric alcohols.

[0101] It is possible where appropriate for the dosage units for oraladministration to be microencapsulated so that delivery is delayed orextended over a longer period, such as, for example, by coating orembedding the active ingredient in particulate form in suitablepolymers, waxes or the like.

[0102] The pharmaceutical products are preferably produced andadministered in dosage units, with each unit containing as activeingredient a particular dose of at least one compound of the formula(I). In the case of solid dosage units such as tablets, capsules andsuppositories, this dose may be up to 1 g, preferably about 0.1 to 200mg, and in the case of injectable solutions in ampul form may be up to 1g, preferably about 0.1 to 100 mg.

[0103] The daily dose to be administered depends on the body weight,age, sex and condition of the mammal. However, higher or lower dailydoses may also be appropriate in some circumstances. Administration ofthe daily dose may take place both by a single administration in theform of a single dosage unit or else in a plurality of smaller dosageunits and by multiple administration of divided doses at particularintervals.

[0104] The medicaments of the invention are produced by converting oneor more compounds of the formula (I) with one or more physiologicallysuitable excipients in a suitable dosage form.

[0105] The invention further relates to the use of a compound of theformula (I) for producing a medicament, in particular for the treatmentof allergic disorders, of asthmatic disorders, of inflammatoryconcomitant symptoms of asthma and/or of diseases which can be treatedby inhibition of c-maf and NFAT.

[0106] The zipper protein c-maf represents the assay target. It plays animportant part in the release of mediators of inflammation, especiallyIL-4, and thus in the manifestation of inflammatory symptoms in asthmaand allergies. c-maf is thus an important therapeutic target moleculefor asthma, especially if it has an allergic cause. The activity ofc-maf is measured in the assay on the basis of the IL-4 transcriptionrate. Compounds which interfere with the binding of the twotranscription factors c-maf and NFAT lead in this case to a reducedexpression of luciferase (read-out) through suppression of thetranscription of a human IL-4 promoter/luciferase construct.

[0107] The following examples are intended to explain the invention inmore detail without wishing to restrict the scope of the invention inany way. Percentage data are based on weight. Mixing ratios of liquidsare based on volume unless stated otherwise.

EXAMPLES Example 1 Plant Production (Collection of the Seeds, Sowing,and Growing and Harvesting Conditions).

[0108] Seeds of Beilschmiedia fulva were collected after ripening andsown to cultivate the plants further in a greenhouse. The optimaltemperature was about 28° C. with a humidity of 70-90%. The plants werecultivated for several months to years until the bark and other suitableparts of the plants were harvested.

Example 2 Preparation of a Primary Extract From Beilschmiedia fulva

[0109] Pieces of Beilschmiedia fulva bark were collected in the freshstate and then dried in air at about 40° C. After drying, 100 g of drymaterial were ground and extracted with 1 l of methanol at 40° C. for 8h while stirring. After the extraction was complete, the plant residueswere filtered off and the methanolic extract was concentrated almost todryness in vacuo. The residue was resuspended again in a little waterand then freeze dried. The primary extract produced in this way could bestored at +4° C. to −20° C. or be used for further isolation as inexample 3. To assay the biological activity, tannins and other stronglyhydrophilic or lipophilic interfering substances were removed from theprimary extract by chromatography on polyamide and on polystyreneadsorber resin.

Example 3 Isolation of Endiandric Acid H From the Plant Beilschmiediafulva

[0110] 100 g of dried pieces of Beilschmiedia fulva bark are harvestedas in example 2, comminuted in a mill, stirred with 1 l of methanol for16 hours and then filtered. The active ingredient-containing methanolicsolution is concentrated in vacuo; the dry matter amounts to 7.0 g. Theconcentrate is loaded onto a prepared glass column (BPG 100, 4 linternal volume, from Pharmacia Biotech) which is packed with about 0.5l of MCI gel® CHP-20P material (adsorber resin from MitsubishiChemicals, Japan). A gradient from 100% water to 100% acetonitrile isused for elution. The column flow-through (50 ml per minute) iscollected in fractions (50 ml each), and fractions active in thebioassay (fraction 18-21) are combined. Concentration in vacuo andfreeze drying afford 102 mg of pale brown powder.

Example 4 Purification of Endiandric Acid H by Reversed Phase HPLC

[0111] The 102 mg of the powder obtained in example 3 were loaded onto aLUNA® 10μ C18 (2) column (size: 21.2 mm×240 mm, from Phenomenex,Germany) and chromatographed with a gradient from 3% to 6% acetonitrilein 0.1% ammonium acetate/water over 60 minutes. The flow-through ofeluent amounts to 33 m/min, and the size of the fractions is 33 ml.Endiandric acid H is present in fractions 24 and 25. Lyophilization ofsaid fractions affords 3.7 mg of pure substance (purity>95%).

Example 5 Characterization of Endiandric Acid H

[0112] Appearance: white substance which is soluble in polar organicsolvents, but only slightly soluble in water.

[0113] UV maxima (in water/acetonitrile): 206, 262, 296.

[0114] The following was found by high-resolution mass spectrometry for(M+H)⁺: 365.1392 amu. This corresponds to a molecular formula ofendiandric acid H of C₂₂H₂₁O₅.

[0115] Electron spray ionization (ESI, positive) results by MS/MSfragmentation in the following ions: 349 amu (M+H—H₂O) to 331 amu(—H₂O), 303 amu (—CH₂O₂), 289 amu (—C₂H₄O₂).

[0116] Electron spray ionization (ESI, negative) results by MS/MSfragmentation in the following ions: 365 amu (M−H)⁻ to 321 amu (—CO₂),267 amu (—C₅H₆O₂) 227 amu (—C₈H₁₀O₂), 215 amu (—C₉H₁₀O₂) and smallerfragments. TABLE 1

NMR chemical shifts and coupling constants of endiandric acid H, DMSO,303 K. δ m δ m Pos. (¹³C) (¹³C) (¹H) (¹H) ^(n) ^(_(J)) CH ^(n) ^(_(J))HH^((J/Hz)) 1 173.47 s — — 2.44, 2.40 — 2 40.69 t 2.440, dd 1.73, 2.351.73 (8.2), 2.40 (12.2) 2.400 dd 1.73 (7.7), 2.44 (12.2) 3 40.90 d 1.733quint 2.77, 2.44, 2.40, 2.44 (8.2), 2.40 (7.70), 1.80, 1.20, (5.59) 2.35(8), 2.34 (8) 4 33.76 d 2.354 5.204, (5.593), 1.73 (8), 2.77 (8), 5.592.765, 2.44, 2.40 (3.5), 5.21 (1) 5 127.59 d 5.593 dt 1.733, 2.765 5.21(10.2), 3.35 (3.5), 2.35 (3.5) 6 126.33 d 5.207 dq 1.851 5.59 (10.2),5.845 (1), 2.35 (1), 3.35 (1) 7 34.82 d 3.351 m 5.20, (5.59), 5.86, 5.85(1), 5.59 (3.5), 5.21 1.85, 1.95 (<1), 4.00 (<1), 2.34 (1), 1.85 (5) 8140.15 s — — 6.91, 7.00, 5.21, — 4.00 9 131.41 d 5.845 q 4.00, (1.95),3.35 (1), 4.00 (1), 5.21 (1.21) (1) 10 71.96 d 4.000 ddd 1.21, 1.85,1.95 1.95 (9), 3.35 (<1), 5.85 (1) 11 44.02 d 1.946 ddt 5.85, 4.00,2.77, 4.00 (9), 1.21 (11.6), 1.21, 1.80, 1.85, 1.80 (4), 1.85 (5) 5.5912 34.99 t 1.801 dd 4.00, 2.76, 1.73 1.21 (11), 1.95 (4) 1.209 dt 2.341.80 (11), 1.95 (11.6), 2.34 (6) 13 40.61 d 2.339 2.44, 2.40, 1.21, 2.77(8), 1.73 (8), 1.80 1.80, 1.73 (<1), 1.21 (6) 14 33.62 d 2.765 q (5.59),2.34, 2.35, 1.85 (8), 2.35 (8), 2.34 1.80, 1.85 (8) 15 40.32 d 1.851 dt1.95, 1.80, 5.21, 2.77 (8), 3.35 (5), 1.95 2.34, (2.77) (5) 1′ 147.51 s— — 6.00, 6.87, (7.00) — 2′ 146.48 s — — 6.00, 7.00, 6.91, — 3′ 108.10 d6.873 d (7.00) 6.91 (8.2) 4′ 119.86 d 6.908 dd 7.00 6.87 (8.2), 7.00(1.6) 5′ 133.76 s — — 6.87, 5.85 — 6′ 106.66 d 6.995 d 6.908 6.81 (1.6)7′ 100.92 t 6.01 s — — 6.00 s

Example 6 Bioassay

[0117] Cell Culture:

[0118] A luciferase reporter gene construct (IL-4 luc) was prepared bymeans of cloning using the human IL-4 promoter (−6635 to +66). The cDNAof full-length murine nuclear factor of activated T cells (N FAT) wascloned into another vector. CHO-K1 cells were then transfected with bothvectors (IL-4 luc/NFAT) by electroporation. It was possible to obtain amonoclonal IL-4 luc/NFAT cell line by a G418 selection process.Subsequently, full-length murine c-maf cDNA was cloned into a suitablevector, and the IL-4 luc/NAFT cell line generated was cotransfectedtherewith. The resulting monoclonal cell line harbors all three vectors(c-maf/IL-4 luc/NFAT) and was used for the screening.

[0119] The cells were cultivated in the logarithmic phase of growth at37° C., 5% CO₂ in tissue culture bottles with an area of 225 cm² in thefollowing medium: Ham's F-12 nutrient mixture supplemented with 10%fetal calf serum, 1% antibiotic/antimycotic, 300 μg/ml Geneticin and 300μg/ml Zeocin.

[0120] Assay Procedure:

[0121] 16 hours before the assay, CHO-K1 cells were harvested bytrypsinization, washed once with PBS without Ca and Mg, resuspended inHam's F-12 medium which additionally contained 10% fetal calf serum, 1%antibiotic/antimycotic, 0.001% Pluronic, 300 μg/ml Geneticin and 300μg/ml Zeocin, and then quantified using a hemocytometer. The cells wereplated out on microtiter plates in a cell concentration of 2 000 cellsper well in 2 μl of medium in each case. The cells are incubated at 37°C. and 5% CO₂ overnight.

[0122] Stock solutions of the assay substances were dissolved in DMSO.These stock solutions were diluted with Ham's F-12 medium whichadditionally contained 10% fetal calf serum, 1% antibiotic/antimycotic(Gibco BRL, No. 15240-062), 300 μg/ml Geneticin and 300 μg/ml Zeocin togive various concentrations. The CHO-K1 cells were mixed with 1 μl ofthe assay substance solutions prepared in this way and then incubated at37° C. and 5% CO₂ for 8 hours. The final concentration of DMSO in thiscase did not exceed 0.83% per well.

[0123] After the incubation time, the cells are in each case mixed with3 μl of Bright-Glow™ luciferase assay reagent (Promega Corporation,Madison, USA) per well. The plates are then placed in the dark for 30minutes and subsequently measured in a CyBiTM Lumax reader. DMSO in afinal concentration of 33.3% was also assayed as positive control. Inaddition, the positive controls were standardized to a concentration of0.83%.

[0124] Evaluation:

[0125] Each assay plate contains 64 negative controls (wells withoutadded substance) and 64 positive controls (wells in which the cells havebeen killed with 1 μl of DMSO).

[0126] The inhibition was calculated as follows:

[0127][1-(LCS_(sample)-LCS_(poscontr))/(LCS_(negcontr)-LCS_(poscontr))]×100(%)

[0128] Endiandric acid H shows an IC₅₀ of 1.5 μM in the bioassay.

What is claimed is:
 1. A compound of the formula (I)

wherein R₁ and R₂ are, independently of one another, 1.0 H or 2.0 a—O—C₁-C₆-alkyl, —O—C₂-C₆-alkenyl, —O—C₂-C₆-alkynyl or —O—C₆-C₁₀-arylgroup, in which alkyl, alkenyl and alkynyl are straight-chain orbranched, and in which the alkyl, alkenyl and alkynyl groups areoptionally mono- or disubstituted by: 2.1 —OH, 2.2 ═O, 2.3—O—C₁-C₆-alkyl in which alkyl is straight-chain or branched, 2.4—O—C₂-C₆-alkenyl in which alkenyl is straight-chain or branched, 2.5—C₆-C₁₀-aryl, 2.6 —NH—C₁-C₆-alkyl in which alkyl is straight-chain orbranched, 2.7 —NH—C₂-C₆-alkenyl in which alkenyl is straight-chain orbranched, 2.8 —NH₂ or 2.9 halogen, and in which the aryl groups areoptionally mono- or disubstituted by substituents 2.1 or 2.3 to 2.9, inwhich the substituents 2.3, 2.4, 2.6 and 2.7 may be further substitutedby —CN, -amide or -oxime functions, and 2.5 may be further substitutedby —CN or amide functions or R₁ and R₂ together form a group—O—[(C₁-C₆)-alkylene]—O—, R₃ is 1.0 H or 2.0 a C₁-C₆-alkyl,C₂-C₆-alkenyl, C₂-C₆-alkynyl or C₆-C₁₀-aryl group, in which alkyl,alkenyl and alkynyl are straight-chain or branched, and in which thealkyl, alkenyl and alkynyl groups are optionally mono- or disubstitutedby: 2.1 —OH, 2.2 =O, 2.3 —O—C₁-C₆-alkyl in which alkyl is straight-chainor branched, 2.4 —O—C₂-C₆-alkenyl in which alkenyl is straight-chain orbranched, 2.5 —C₆-C₁₀-aryl, 2.6 —NH—C₁-C₆-alkyl in which alkyl isstraight-chain or branched, 2.7 —NH—C₂-C₆-alkenyl in which alkenyl isstraight-chain or branched, 2.8 —NH₂ or 2.9 halogen, and in which thearyl groups are optionally mono- or disubstituted by substituents 2.1 or2.3 to 2.9, in which the substituents 2.3, 2.4, 2.6 and 2.7 may befurther substituted by —CN, -amide or -oxime functions, and 2.5 may befurther substituted —CN or amide, and R₄ is CO₂R₃, CO₂NHR₃, CHO, CH₂OR₃,CH₂OSi(R₃)₃, CH₂Br, CH₂CN, where R₃ is as defined above, or astereoisomeric form of the compound of the formula (I) or aphysiologically tolerated salt of the compound of the formula (I) or asalt of a stereoisomeric form of the compound of the formula(l).
 2. Thecompound according to claim 1, which is the compound of formula (II)


3. The compound according to claim 1, which is the compound of formula(III)


4. The compound according to claim 1, which is the compound of formula(IV)


5. A process for the preparation of the compound of formula (I),according to claim 1 comprising:
 1. extracting the plant Beilschmiediafulva, PLA 101037, or cell cultures of the plant Beilschmiedia fulva,PLA 101037, under suitable conditions,
 2. isolating the compound of theformula (IV),


3. where appropriate derivatizing to a compound of the formula (I)and/or reacting to give a physiologically tolerated salt of the compoundof the formula (I).
 6. A process for the preparation of the compound offormula (IV) according to claim 4 comprising:
 1. extracting the plantBeilschmiedia fulva, PLA 101037, or cell cultures of the plantBeilschmiedia fulva, PLA 101037, under suitable conditions,
 2. isolatingthe compound of the formula (IV), and
 3. where appropriate reacting togive a physiologically tolerated salt of the compound of the formula(IV).
 7. A pharmaceutical composition comprising a compound of claim 1or a pharmacologically tolerable salt thereof and one or morephysiologically acceptable excipients.
 8. A process for the preparationof a pharmaceutical composition as claimed in claim 7, comprisingbringing a compound of formula (I), or a pharmacologically tolerablesalt thereof, into a suitable administration form using one or morephysiologically suitable excipients.
 9. A method of inhibiting c-maf andNFAT to treat a disease or disorder which comprises administering to apatient in need of said treatment or disorder an effective c-maf andNFAT inhibitory amount of a compound according to claim
 1. 10. Themethod according to claim 9 wherein the disease or disorder is selectedfrom the group consisting of allergies, asthma and inflammatory symptomsassociated with asthma.